The present invention is related to results obtained from research on human deoxyribonuclease I (DNase I), a phosphodiesterase that is capable of hydrolyzing polydeoxyribonucleic acid. It relates generally to modified (variant) forms of human DNase I having increased DNA-hydrolytic activity and their preparation by recombinant DNA methods, to pharmaceutical compositions by which their utility can be exploited clinically, and to methods of using these DNase I variants and compositions thereof.
DNase I is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. DNase I has been purified from various species to various degrees.
Bovine DNase I has been extensively studied biochemically. See e.g., Moore, in The Enzymes (Boyer, P. D., ed), pp. 281-296, Academic press, New York (1981). The complete amino acid sequence for bovine DNase I is known (Liao, et al., J. Biol. Chem. 248:1489-1495 (1973); Oefner, et al., J. Mol. Biol. 192:605-632 (1986); Lahm, et al., J. Mol. Biol. 221:645-667 (1991)), and DNA encoding bovine DNase I has been cloned and expressed (Worrall, et al., J. Biol. Chem 265:21889-21895 (1990)). The structure of bovine DNase I has been determined by X-ray crystallography. Suck, et al., EMBO J. 3:2423-2430 (1984); Suck, et al., Nature 321:620-625 (1986); Oefner, et al., J. Mol. Biol. 192:605-632 (1986); Weston, et al., J. Mol. Biol. 226:1237-1256 (1992).
DNA encoding human DNase I has been isolated and sequenced and that DNA has been expressed in recombinant host cells, thereby enabling the production of human DNase I in commercially useful quantities. Shak, et al., Proc. Nat. Acad. Sci. 87:9188-9192 (1990).
DNase I has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions (mucus) in such diseases as pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of respiratory airways. See e.g., Lourenco, et al., Arch. Intern. Med. 142:2299-2308 (1982); Shak, et al., Proc. Nat. Acad. Sci. 87:9188-9192 (1990); Hubbard, et al., New Engl. J. Med. 326:812-815 (1992); Fuchs, et al., New Engl. G. Med. 331:637-642 (1994); Bryson, et al., Drugs 48:894-906 (1994). Mucus also contributes to the morbidity of chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and even the common cold.
The pulmonary secretions of persons having such diseases are complex materials, that include mucus glycoproteins, mucopolysaccharides, proteases, actin, and DNA. Some of the materials in pulmonary secretions are released from leukocytes (neutrophils) that infiltrate pulmonary tissue in response to the presence of microbes (e.g., strains of Pseudomonas, Pneumococcus, or Staphylococcus bacteria) or other irritants (e.g., tobacco smoke, pollen). In the course of reacting with-such microbes or irritants, the leukocytes may degenerate and release their contents, which contribute to the viscoelasticity of the pulmonary secretions.
The ability of DNase I to reduce the viscoelasticity of pulmonary secretions has been ascribed to its enzymatic degradation of the large amounts of DNA released by neutrophils. Shak, et al., Proc. Nat. Acad. Sci. 87:9188-9192 (1990); Aitken, et al., J. Am. Med. Assoc. 267:1947-1951 (1992).
The present invention is based in part on research by the inventors to study the enzymatic activity of human DNase I. This research involved the design and synthesis of various human DNase I variants, and the assay of these variants to assess their ability to hydrolyze DNA in vitro. The inventors have identified for the first time a class of human DNase I variants, termed hyperactive variants, that have increased DNA-hydrolytic activity and that are less susceptible to inhibition by sodium chloride, as compared to native human DNase I.
Because of their increased DNA-hydrolytic activity, the hyperactive variants also have increased mucolytic activity and are more effective than native human DNase I in degrading (digesting) DNA generally. Because they are less susceptible to inhibition by sodium chloride, the hyperactive variants are more effective than native human DNase I under physiological saline conditions, such as occur in pulmonary secretions and other biological fluids. Accordingly, hyperactive variants of human DNase I should be especially useful in treating patients having pulmonary secretions that comprise relatively large amounts of DNA.
It is therefore an object of the present invention to provide human DNase I variants that have greater DNA-hydrolytic activity than native human DNase I.
It is another object of the invention to provide nucleic acids encoding such hyperactive variants of human DNase I, recombinant vectors comprising such nucleic acids, recombinant host cells transformed with those nucleic acids or vectors, and processes for producing the human DNase I variants by means of recombinant DNA technology. The invention includes the use of such nucleic acids and vectors for in vivo or ex vivo gene therapy.
The invention also is directed to pharmaceutical compositions comprising the hyperactive variants of human DNase I, optionally together with a pharmaceutically acceptable excipient, as well as substantially purified antibodies that are capable of binding to such hyperactive variants.
The invention also is directed to methods of use of the hyperactive variants. Included are methods for reducing the viscoelasticity or viscous consistency of DNA-containing material in a patient, and for reducing or preventing formation of DNA-containing immune complexes in a patient, comprising administering a therapeutically effective dose of a hyperactive variant of human DNase I to the patient.
The invention is particularly directed to a method of treating a patient having a disease such as cystic fibrosis, chronic bronchitis, pneumonia, bronchiectasis, emphysema, asthma, or systemic lupus erythematosus, that comprises administering a therapeutically effective amount of a hyperactive variant of human DNase I to the patient.
These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole.